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1.
Medicine (Baltimore) ; 99(4): e18543, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31977847

RESUMO

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer with a high mortality disease which has been positioned the first and second cancer morbidity of men and women in China, separately. Our study was to assess the prognostic meaningful of ubiquitin conjugating enzyme E2 T (UBE2T) expression in LUAD dependent on data acquired from The Cancer Genome Atlas (TCGA) and so as to increase further knowledge into the biological pathways involved in LUAD pathogenesis related to UBE2T.Information on gene expression and comparing clinical data were recognized and downloaded from TCGA. Gene set enrichment analysis (GSEA) created an arranged list of all genes s indicated by their connection with UBE2T expression.Our study cohort included 265 (54.5%) female and 221 (36.0%) male patients. The scatter plot and paired plot showed the difference of UBE2T expression between normal and tumor samples (P < .01). Overall survival (OS) analysis demonstrated that LUAD with UBE2T-high had a more terrible prognosis than that with UBE2T-low (P < .01). Multivariate analysis with the cox proportional hazards model indicated that the expression of UBE2T (hazard ratio [HR]: 1.28; 95% Confidence Interval (CI): 1.06-1.56; P = .011) and stage (HR: 2.02; 95% CI: 1.27-3.21; P = .003) were independent prognostic factors for patients with LUAD. The GSEA results showed that cell cycle, DNA replication, RNA degradation, oxidative phosphorylation, pathogenic Escherichia coli infection, citrate cycle tricarboxylic acid cycle, Alzheimer's disease, P53 signaling pathway, and purine metabolism are differentially enriched in UBE2T high expression phenotype.Our study found that the expression of UBE2T was significantly increased in LUAD patients and associated with several clinical features. UBE2T may be a potentially useful prognostic molecular biomarker of bad survival in LUAD, while further experimental ought to be performed to demonstrate the biologic effect of UBE2T.


Assuntos
Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/patologia , RNA Mensageiro/biossíntese , Enzimas de Conjugação de Ubiquitina/biossíntese , Adenocarcinoma de Pulmão/mortalidade , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Ciclo Celular , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fenótipo , Prognóstico , Fatores de Risco , Fatores Sexuais , Análise de Sobrevida
2.
Redox Biol ; 28: 101364, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731101

RESUMO

Inflammation is a self-defense response to protect individuals from infection and tissue damage, but excessive or persistent inflammation can have adverse effects on cell survival. Many individuals become especially susceptible to chronic-inflammation-induced sensorineural hearing loss as they age, but the intrinsic molecular mechanism behind aging individuals' increased risk of hearing loss remains unclear. FoxG1 (forkhead box transcription factor G1) is a key transcription factor that plays important roles in hair cell survival through the regulation of mitochondrial function, but how the function of FoxG1 changes during aging and under inflammatory conditions is unknown. In this study, we first found that FoxG1 expression and autophagy both increased gradually in the low concentration lipopolysaccharide (LPS)-induced inflammation model, while after high concentration of LPS treatment both FoxG1 expression and autophagy levels decreased as the concentration of LPS increased. We then used siRNA to downregulate Foxg1 expression in hair cell-like OC-1 cells and found that cell death and apoptosis were significantly increased after LPS injury. Furthermore, we used d-galactose (D-gal) to create an aging model with hair cell-like OC-1 cells and cochlear explant cultures in vitro and found that the expression of Foxg1 and the level of autophagy were both decreased after D-gal and LPS co-treatment. Lastly, we knocked down the expression of Foxg1 under aged inflammation conditions and found increased numbers of dead and apoptotic cells. Together these results suggest that FoxG1 affects the sensitivity of mimetic aging hair cells to inflammation by regulating autophagy pathways.

3.
Eur J Pharmacol ; 853: 74-83, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30880181

RESUMO

The inhibition of transient outward potassium current (Ito) is the major ionic mechanism for quinidine to treat Brugada syndrome; however, quinidine is inaccessible in many countries. The present study compared the inhibitory effect of the nonselective ß-adrenergic blocker carvedilol with quinidine on human Kv4.3 (hKv4.3, encoding for Ito) channel and action potential notch using a whole-cell patch technique in HEK 293 cell line expressing KCND3 as well as in ventricular epicardial myocytes of rabbit hearts. It was found that carvedilol and quinidine inhibited hKv4.3 current in a concentration-dependent manner. The IC50 of carvedilol was 1.2 µM for inhibiting hKv4.3 charge area, while the IC50 of quinidine was 2.9 µM (0.2 Hz). Both carvedilol and quinidine showed typical open channel blocking properties (i.e. decreasing the time to peak of activation and increasing the inactivation of hKv4.3), negatively shifted the V1/2 of activation and inactivation, and slowed the recovery from inactivation of the channel. Although carvedilol had weaker in use- and rate-dependent inhibition of hKv4.3 peak current than quinidine, its reduction of the charge area was more than quinidine at all frequencies (0.2-3.3 Hz). Moreover, the inhibitory effect of carvedilol on action potential notch was greater than quinidine. These results provide the novel information that carvedilol, like quinidine, significantly inhibits hKv4.3 and action potential notch, suggesting that carvedilol is likely an alternative drug for preventing malignant ventricular arrhythmias in patients with Brugada syndrome in countries where quinidine is unavailable.


Assuntos
Carvedilol/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Quinidina/farmacologia , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Expressão Gênica , Células HEK293 , Ventrículos do Coração/citologia , Humanos , Concentração Inibidora 50 , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Coelhos , Canais de Potássio Shal/metabolismo
4.
Br J Pharmacol ; 175(16): 3422-3432, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29900525

RESUMO

BACKGROUND AND PURPOSE: Adrenergic regulation of cell volume-regulated chloride current (ICl.vol ) is species-dependent. The present study investigates the mechanism underlying adrenergic regulation of ICl.vol in human atrial myocytes. EXPERIMENTAL APPROACH: Conventional whole-cell patch voltage-clamp techniques were used to record membrane current in human atrial myocytes. ICl.vol was evoked by hyposmotic bath solution (0.6 times isosmotic, 0.6 T). KEY RESULTS: ICl.vol was augmented by noradrenaline (1 µM) during cell swelling in 0.6 T but not under isosmotic (1 T) conditions. Up-regulation of ICl.vol in 0.6 T was blocked by the ß-adrenoceptor antagonist propranolol (2 µM), but not by the α1 -adrenoceptor antagonist prazosin (2 µM). This ß-adrenergic response involved cAMP but was independent of PKA; the protein kinase inhibitor H-89 (2 µM) or PKI (10 µM in pipette solution) failed to prevent ICl.vol up-regulation by noradrenaline. Moreover, the PI3K/PKB inhibitor LY294002 (50 µM) and the PKG inhibitor KT5823 (10 µM) did not affect noradrenaline-induced increases in ICl.vol . Interestingly, the exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2'-O-Me-cAMP (50 µM) also up-regulated ICl.vol , and the noradrenaline-induced increase of ICl.vol in 0.6 T was reversed or prevented by the Epac inhibitor ESI-09 (10 µM). CONCLUSION AND IMPLICATIONS: These data show that ICl.vol evoked by cell swelling of human atrial myocytes is up-regulated by noradrenaline via a PKA-independent cAMP/Epac pathway in human atrial myocytes. cAMP/Epac-induced ICl.vol is expected to shorten action potential duration during human atrial myocytes swelling and may be involved in abnormal cardiac electrical activity during cardiac pathologies that evoke ß-adrenoceptor signalling.


Assuntos
AMP Cíclico/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Norepinefrina/farmacologia , Células Cultivadas , Cloretos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico , Átrios do Coração/citologia , Humanos , Miócitos Cardíacos/fisiologia , Regulação para Cima/efeitos dos fármacos
6.
Front Pharmacol ; 8: 716, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29081746

RESUMO

The natural flavone acacetin inhibits several voltage-gated potassium currents in atrial myocytes, and has anti-atrial fibrillation (AF) effect in experimental AF models. The present study investigates whether acacetin inhibits the Ca2+-activated potassium (KCa) currents, including small conductance (SKCa1, SKCa2, and SKCa3), intermediate conductance (IKCa), and large-conductance (BKCa) channels stably expressed in HEK 293 cells. The effects of acacetin on these KCa channels were determined with a whole-cell patch voltage-clamp technique. The results showed that acacetin inhibited the three subtype SKCa channel currents in concentration-dependent manner with IC50 of 12.4 µM for SKCa1, 10.8 µM for SKCa2, and 11.6 µM for SKCa3. Site-directed mutagenesis of SKCa3 channels generated the mutants H490N, S512T, H521N, and A537V. Acacetin inhibited the mutants with IC50 of 118.5 µM for H490N, 275.2 µM for S512T, 15.3 µM for H521N, and 10.6 µM for A537V, suggesting that acacetin interacts with the P-loop helix of SKCa3 channel. However, acacetin at 3-10 µM did not decrease, but induced a slight increase of BKCa (+70 mV) by 8% at 30 µM. These results demonstrate the novel information that acacetin remarkably inhibits SKCa channels, but not IKCa or BKCa channels, which suggests that blockade of SKCa by acacetin likely contributes to its anti-AF property previously observed in experimental AF.

7.
FEBS Open Bio ; 7(6): 759-776, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28593132

RESUMO

Degeneration of the central auditory system, which is characterized by reduced understanding of speech and source localization of sounds, is an important cause of age-related hearing loss (presbycusis). Accumulating evidence has demonstrated that Wnt/ß-catenin signaling plays an essential role in the development of the auditory system but its potential role in presbycusis remains unclear. In this study, we used a rat model of aging, created by chronic systemic exposure to d-galactose (d-gal), and explored changes in Wnt/ß-catenin signaling in the auditory cortex. A decrease in Wnt/ß-catenin signaling in the auditory cortex was found in both naturally aging and d-gal-mimetic aging rats, as indicated by increased GSK3ß activity and decreased ß-catenin activity. Moreover, lithium chloride (Licl), an activator of Wnt signaling pathway, was administered long term to 15-month-old d-gal-treated rats. Activation of Wnt/ß-catenin signaling by Licl attenuated d-gal-induced auditory cortex apoptosis and neurodegeneration. Bmi1, a transcription factor implicated in antiaging and resistance to apoptosis, can be modulated by ß-catenin activity. Here, we showed that the expression of Bmi1 was reduced and the expression of its downstream genes, p16INK4a , p19Arf , and p53 were increased in the auditory cortex both of naturally aging and d-gal-mimetic aging rats. In addition, Licl significantly increased Bmi1 expression and reduced p16INK4a, p19Arf, and p53 expression. Our results indicated that decreased Wnt/ß-catenin signaling might participate in the pathogenesis of central presbycusis through modulating the expression of Bmi1. Wnt/ß-catenin signaling might be used as a potential therapeutic target against presbycusis.

8.
Free Radic Res ; 51(5): 517-528, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28482716

RESUMO

Dihydromyricetin (DHM), a Rattan tea extract, has recently been shown to have anti-cancer activity in mammalian cells. In this study, we investigated the effect of DHM on human melanoma cells. Apart from induction of apoptosis, we demonstrated that DHM induced an autophagic response. Moreover, pharmacological inhibition or genetic blockade of autophagy enhanced DHM-induced cell death and apoptosis, indicating the cytoprotective role of autophagy in DHM-treated human melanoma cells. Further study suggested that the nuclear factor kappa B (NF-κB) signalling pathway was involved in DHM-induced autophagy. Moreover, N-acetyl-cysteine (NAC), an ROS scavenger, abrogated the effects of DHM on NF-κB-dependent autophagy. Taken together, this evidence demonstrates that a strategy of blocking ROS-NF-κB-dependent autophagy to enhance the activity of DHM warrants further attention for the treatment of human melanoma.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Flavonóis/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Autofagia , Linhagem Celular Tumoral , Citoproteção , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
9.
ORL J Otorhinolaryngol Relat Spec ; 79(3): 153-163, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28407635

RESUMO

BACKGROUND/AIMS: According to recent studies, central auditory impairments are closely related to neurodegenerative diseases. However, the mechanism of central presbycusis remains unclear. Ubiquitin C-terminal hydrolase L1 (UCHL1) is important in maintaining proteasomal activity; however, the detailed mechanism has not yet been fully elucidated. This study aims to investigate the molecular alterations involved in UCHL1 regulation during auditory cortex aging. METHODS: D-Galactose (D-gal) induces oxidative stress and senescence in the auditory cortex, as reported in our previous studies. Primary auditory cortex cells were treated with D-gal for 72 h or 5 days. The proteins related to the ubiquitin proteasome system (UPS) and proteasomal activities were evaluated. UCHL1 was overexpressed, and the effects of UCHL1 on the UPS and proteasomal activity were analyzed. RESULTS: Proteasomal activity was elevated at 72 h and decreased at 5 days in D-gal-treated primary auditory cortex cells. We also found that overexpression of UCHL1 increased the UPS-related proteins UBE1, PSMA7, ubiquitinated proteins, and monoubiquitin, and proteasomal activity. CONCLUSION: The results suggest that UCHL1 may modify the aging process in the auditory cortex by regulating UPS- related proteins.


Assuntos
Envelhecimento/metabolismo , Córtex Auditivo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina Tiolesterase/metabolismo , Análise de Variância , Animais , Biomarcadores/análise , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Papel (figurativo) , Sensibilidade e Especificidade
10.
J Cell Mol Med ; 21(9): 1826-1834, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28294531

RESUMO

The present study was designed to investigate whether large conductance Ca2+ -activated K+ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK-HEK 293 cells expressing both functional α-subunits and the auxiliary ß1-subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK-HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α- and ß1-subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre-contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.


Assuntos
Receptores ErbB/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Tirfostinas/farmacologia , Animais , Artéria Basilar/citologia , Separação Celular , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Genisteína/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Subunidades Proteicas/metabolismo , Ratos Sprague-Dawley , Vanadatos/farmacologia , Vasodilatação/efeitos dos fármacos
11.
Br J Pharmacol ; 174(6): 454-467, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28072464

RESUMO

BACKGROUND AND PURPOSE: The ultra-rapidly activating delayed rectifier K+ current IKur (encoded by Kv 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). EXPERIMENTAL APPROACH: Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial IKur and hKv1.5 channels stably expressed in HEK 293 cells. KEY RESULTS: Human atrial IKur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of IKur induced by 30 µM genistein or 10 µM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hKv 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 µM) slightly enhanced IKur and hKv 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hKv 1.5 channels and that the decrease was countered by orthovanadate. CONCLUSION AND IMPLICATIONS: The PTK inhibitors genistein and AG556 decrease human atrial IKur and cloned hKv 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases IKur and hKv 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial IKur .


Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Genisteína/farmacologia , Átrios do Coração/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tirfostinas/farmacologia , Células Cultivadas , Canais de Potássio de Retificação Tardia/metabolismo , Receptores ErbB/metabolismo , Genisteína/química , Células HEK293 , Átrios do Coração/metabolismo , Humanos , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Tirfostinas/química
12.
Sci Rep ; 6: 36435, 2016 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-27819271

RESUMO

The morbidity and mortality of patients with ischemic cardiomyopathy resulted from ischemia/reperfusion injury are very high. The present study investigates whether our previously synthesized water-soluble phosphate prodrug of acacetin was cardioprotective against ischemia/reperfusion injury in an in vivo rat model. We found that intravenous administration of acacetin prodrug (10 mg/kg) decreased the ventricular arrhythmia score and duration, reduced ventricular fibrillation and infarct size, and improved the impaired heart function induced by myocardial ischemia/reperfusion injury in anesthetized rats. The cardioprotective effects were further confirmed with the parent compound acacetin in an ex vivo rat regional ischemia/reperfusion heart model. Molecular mechanism analysis revealed that acacetin prevented the ischemia/reperfusion-induced reduction of the anti-oxidative proteins SOD-2 and thioredoxin, suppressed the release of inflammation cytokines TLR4, IL-6 and TNFα, and decreased myocyte apoptosis induced by ischemia/reperfusion. Our results demonstrate the novel evidence that acacetin prodrug confer significant in vivo cardioprotective effect against ischemia/reperfusion injury by preventing the reduction of endogenous anti-oxidants and the release of inflammatory cytokines, thereby inhibiting cardiomyocytes apoptosis, which suggests that the water-soluble acacetin prodrug is likely useful in the future as a new drug candidate for treating patients with acute coronary syndrome.


Assuntos
Flavonas/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Pró-Fármacos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Flavonas/química , Flavonas/metabolismo , Flavonas/farmacologia , Coração/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Interleucina-6/metabolismo , Masculino , Modelos Biológicos , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Substâncias Protetoras/química , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Pressão Ventricular/efeitos dos fármacos
13.
Sci Rep ; 6: 25743, 2016 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-27160397

RESUMO

We previously reported that duodenal administration of the natural flavone acacetin can effectively prevent the induction of experimental atrial fibrillation (AF) in canines; however, it may not be used intravenously to terminate AF due to its poor water-solubility. The present study was to design a water-soluble prodrug of acacetin and investigate its anti-AF effect in beagle dogs. Acacetin prodrug was synthesized by a three-step procedure. Aqueous solubility, bioconversion and anti-AF efficacy of acacetin prodrug were determined with different methodologies. Our results demonstrated that the synthesized phosphate sodium salt of acacetin prodrug had a remarkable increase of aqueous solubility in H2O and clinically acceptable solution (5% glucose or 0.9% NaCl). The acacetin prodrug was effectively converted into acacetin in ex vivo rat plasma and liver microsome, and in vivo beagle dogs. Intravenous infusion of acacetin prodrug (3, 6 and 12 mg/kg) terminated experimental AF without increasing ECG QTc interval in beagle dogs. The intravenous LD50 of acacetin prodrug was 721 mg/kg in mice. Our preclinical study indicates that the synthesized acacetin prodrug is highly water-soluble and safe; it effectively terminates experimental AF in beagle dogs and therefore may be a promising drug candidate for clinical trial to treat patients with acute AF.


Assuntos
Fibrilação Atrial/tratamento farmacológico , Flavonas/síntese química , Flavonas/uso terapêutico , Pró-Fármacos/síntese química , Pró-Fármacos/uso terapêutico , Água/química , Animais , Fibrilação Atrial/sangue , Cães , Flavonas/sangue , Flavonas/farmacocinética , Humanos , Camundongos Endogâmicos ICR , Canais de Potássio/metabolismo , Pró-Fármacos/farmacocinética , Ratos , Solubilidade , Testes de Toxicidade Aguda , Nervo Vago/efeitos dos fármacos
14.
J Cell Mol Med ; 20(6): 1118-27, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26865051

RESUMO

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells.


Assuntos
Miocárdio/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/metabolismo , Canais de Cátion TRPV/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Movimento Celular , Proliferação de Células , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Espaço Intracelular/metabolismo , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Canais de Cátion TRPV/genética
15.
Pharmacol Res ; 104: 61-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26689773

RESUMO

SKF-96365 is a TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in different systems, which was recently reported to induce QTc prolongation on ECG by inhibiting TRPC channels. The present study investigates whether the blockade of cardiac repolarization currents would be involved in the increase of QTc interval. Cardiac repolarization currents were recorded in HEK 293 cells stably expressing human ether-à-go-go-related gene potassium (hERG or hKv11.1) channels, hKCNQ1/hKCNE1 channels (IKs) or hKir2.1 channels and cardiac action potentials were recorded in guinea pig ventricular myocytes using a whole-cell patch technique. The potential effect of SKF-96365 on QT interval was evaluated in ex vivo guinea pig hearts. It was found that SKF-96365 inhibited hERG current in a concentration-dependent manner (IC50, 3.4µM). The hERG mutants S631A in the pore helix and F656V of the S6 region reduced the inhibitory sensitivity with IC50s of 27.4µM and 11.0µM, suggesting a channel pore blocker. In addition, this compound inhibited IKs and hKir2.1currents with IC50s of 10.8 and 8.7µM. SKF-96365 (10µM) significantly prolonged ventricular APD90 in guinea pig ventricular myocytes and QTc interval in ex vivo guinea pig hearts. These results indicate that the TRPC channel antagonist SKF-96365 exerts blocking effects on hERG, IKs, and hKir2.1 channels. Prolongation of ventricular APD and QT interval is related to the inhibition of multiple repolarization potassium currents, especially hERG channels.


Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Imidazóis/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Eletrocardiografia/efeitos dos fármacos , Cobaias , Células HEK293 , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia
16.
Biol Blood Marrow Transplant ; 22(2): 212-219, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26555814

RESUMO

Numerous previous studies have suggested that cytotoxic T lymphocyte antigen-4 (CTLA-4) plays an important role in acute graft-versus-host disease (GVHD). How CTLA-4 acts in regulating acute GVHD remains unknown, however. In the present study, we found that, compared with healthy controls, CTLA-4 plasma and relative mRNA levels in patients with acute GVHD were initially decreased and then markedly elevated after 28 days of treatment. CTLA-4 levels were higher in patients with grade I-II acute GVHD compared with those with grade III-IV acute GVHD both before and after treatment. Up-regulation of CTLA-4 significantly increased the luciferase activity and degree of phosphorylation of signal transducer and activator of transcription 3 (STAT3). Meanwhile, T cell activation was significantly inhibited, and levels of IFN-γ, IL-17, and IL-22 decreased. These findings suggest that CTLA-4 might be involved in the pathogenesis of acute GVHD, and may down-regulate T helper 1 cells by increasing STAT3 expression in acute GVHD.


Assuntos
Antígeno CTLA-4/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Adolescente , Adulto , Feminino , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Adulto Jovem
17.
Heart Rhythm ; 13(3): 762-70, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26598320

RESUMO

BACKGROUND: Several mammalian species display distinct biophysical properties between atrial and ventricular voltage-gated sodium current (INa); however, the potential mechanism behind this phenomenon is unknown. OBJECTIVE: The purpose of this study was to investigate the potential molecular identities of the different INa in atrial and ventricular myocytes of rat hearts. METHODS: Whole-cell patch voltage-clamp and molecular biology techniques were used in the study. RESULTS: Ventricular INa exhibited a slower inactivation, more positive potential of inactivation, and quicker recovery from inactivation compared to atrial INa. Real-time polymerase chain reaction and western blot analysis revealed that mRNA and protein levels of NaVß2 and NaVß4 subunits, but not NaV1.5, were greater in ventricular myocytes than in atrial myocytes. INa in heterologous HEK 293 cell expression system with coexpressing hNaV1.5 and hNaVß2/hNaVß4 showed similar biophysical properties to ventricular INa. Greater protein expression of NaVß2 and NaVß4 subunits was also observed in human ventricles. Interestingly, pharmacologic study revealed that the antiarrhythmic drug dronedarone (10 µM) inhibited atrial INa more (by 73%) than ventricular INa (by 42%), and shifted its inactivation to more negative voltages (-4.6 mV) compared to ventricular INa. CONCLUSION: The results of this study demonstrate the novel information that the distinctive biophysical properties of INa in atrial and ventricular myocytes can be attributed to inhomogeneous expression of NaVß2 and NaVß4 subunits, and that atrial INa is more sensitive to inhibition by dronedarone.


Assuntos
Amiodarona/análogos & derivados , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Amiodarona/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Dronedarona , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/patologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Técnicas de Patch-Clamp , Ratos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos
18.
Am J Physiol Heart Circ Physiol ; 309(10): H1772-81, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26453325

RESUMO

Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.


Assuntos
Canais de Cálcio/genética , Sinalização do Cálcio/genética , Ciclo Celular/genética , Movimento Celular/genética , Miocárdio/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Células-Tronco/metabolismo , Ensaios de Migração Celular , Proliferação de Células/genética , Células Cultivadas , Ciclina D1/metabolismo , Ciclina E/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteína ORAI1 , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Interação Estromal , Canais de Cátion TRPC/genética
19.
PLoS One ; 10(9): e0138581, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26390131

RESUMO

Our previous study demonstrated that a large-conductance Ca2+-activated K+ current (BKCa), a voltage-gated TTX-sensitive sodium current (INa.TTX), and an inward rectifier K+ current (IKir) were heterogeneously present in most of human cardiac c-kit+ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit+ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BKCa with paxilline, but not INa.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of IKir with Ba2+ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BKCa channels, but not INa.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit+ progenitor cells.


Assuntos
Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Células-Tronco/metabolismo , Bário/farmacologia , Western Blotting , Ciclo Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Citometria de Fluxo , Humanos , Indóis/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Miocárdio/citologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
20.
Thromb Res ; 136(4): 797-802, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26272303

RESUMO

INTRODUCTION: Previously, we demonstrated the importance of T-cell immune response cDNA 7 (TIRC7) in acute immune thrombocytopenia (ITP). As the downstream molecule of TIRC7, cytotoxic T lymphocyte antigen-4 (CTLA-4) has been verified its negative regulation of acute ITP. This study aimed to investigate the exact role of CTLA-4 and its relationship with TIRC7 in acute ITP. PATIENTS AND METHODS: 37 patients with acute ITP were enrolled and received dexamethasone (40mg/day) for 4 consecutive days. Patients who had platelet counts more than 50×10(9)/L or less were defined as responders or non-responders after treatment. The plasma, protein and mRNA levels of CTLA-4 and TIRC7 were monitored by ELISA, western blot and q-PCR, respectively. RESULTS: After high-dose dexamethasone therapy, CTLA-4 levels were significantly elevated not only in acute ITP patients (P<0.001; P<0.0001) but also in acute ITP responders (P<0.0001; P<0.0001). The levels of CTLA-4 were negatively correlated with the levels of TIRC7 before and after treatment; IFN-γ (Th1), IL-17 (Th17) and IL-22 (Th22) levels were all elevated, which were decreased after treatment not only in patients with acute ITP (P<0.01) but also in acute ITP responders (P<0.01). CONCLUSIONS: CTLA-4 level might reflect treatment efficacy and it might be associated with the pathogenesis of acute ITP.


Assuntos
Antígeno CTLA-4/sangue , Púrpura Trombocitopênica Idiopática/sangue , Linfócitos T Citotóxicos/metabolismo , Adolescente , Adulto , Antígeno CTLA-4/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Idiopática/imunologia , Linfócitos T Citotóxicos/imunologia , ATPases Vacuolares Próton-Translocadoras/sangue , ATPases Vacuolares Próton-Translocadoras/imunologia , Adulto Jovem
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